Proper positioning of the mitotic spindle is essential for a number of developmental processes, including asymmetric divisions in which a polarized cell divides to produce daughters with different fates. This project addresses the mechanisms by which the conserved PAR polarity proteins and a downstream intermediate, LET-99, regulate spindle position during asymmetric divisions in the model organism Caenorhabditis elegans. Several hypotheses about LET-99 function will be tested. In Aim 1, the genetic pathway by which the PAR proteins localize LET-99 in a cortical band will be determined by examining LET-99 localization in mutant backgrounds. In addition, the regions of the LET-99 protein that are necessary and sufficient for cortical and asymmetric localization will be identified using transgenes. In Aim 2, the hypothesis that the cortical LET-99 band controls spindle positioning will be tested by time-lapse video microscopy of mutants with altered LET-99 distributions. Aim 3 will test the hypothesis that LET-99 antagonizes the G protein signaling pathway that functions in spindle positioning. Genetic interactions will be examined and possible associations between LET-99 and components of the G protein pathway will be assayed using co-immunoprecipitations and two-hybrid assays. In Aim 4, the hypothesis that the spn-2 gene acts with LET-99 to antagonize G protein signaling will be tested. In the long term, these studies will lead to an in-depth understanding of the molecular mechanisms that coordinate polarity and spindle orientation. Asymmetric divisions are important to the development of all multcellular organisms, and the PAR proteins are conserved in many organisms, including humans. Thus, insights gained from this work will be of wide relevance and could also aid in the understanding of developmental abnormalities that lead to cancer or other disorders. [unreadable] [unreadable]